1. Field of the Invention
The invention includes methods of inhibiting, inhibitors and methods of discovery of inhibitors of HIV infection.
2. Background
Human Immunodeficiency Virus (HIV) is a member of the lentiviruses, a subfamily of retroviruses. Many retroviruses are well-known carcinogens. HIV per se is not known to cause cancer in humans or other animals, but it does present a formidable challenge to the host. The viral genome contains many regulatory elements which allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system; it breaks down the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4, also known as OKT4, T4 and leu3. The viral tropism is due to the interactions between the viral envelope glycoprotein, gp120, and the cell-surface CD4 molecules (Dalgleish et al., Nature 312:763-767 (1984)). These interactions not only mediate the infection of susceptible cells by HIV, but are also responsible for the virus-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in HIV-infected patients. These events result in HIV-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
In addition to CD4+ T cells, the host range of HIV includes cells of the mononuclear phagocytic lineage (Dalgleish et al., supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage and monocytes are major reservoirs of HIV. They can interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
Considerable progress has been made in the development of drugs for HIV-1 therapy. Therapeutic agents for HIV can include, but not are not limited to, at least one of AZT, 3TC, ddC, d4T, ddI, tenofovir, abacavir, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, lopinavir and amprenavir, or any other antiretroviral drugs or antibodies in combination with each other, or associated with a biologically based therapeutic, such as, for example, gp41-derived peptides enfuvirtide (Fuzeon; Timeris-Roche) and T-1249 (Trimeris), or soluble CD4, antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionally presented herein. Combinations of these drugs are particularly effective and can reduce levels of viral RNA to undetectable levels in the plasma and slow the development of viral resistance, with resulting improvements in patient health and life span.
Despite these advances, there are still problems with the currently available drug regimens. Many of the drugs exhibit severe toxicities, have other side-effects (e.g., fat redistribution) or require complicated dosing schedules that reduce compliance and thereby limit efficacy. Resistant strains of HIV often appear over extended periods of time even on combination therapy. The high cost of these drugs is also a limitation to their widespread use, especially outside of developed countries.
There is still a major need for the development of additional drugs to circumvent these issues. Ideally these would target different stages in the viral life cycle, adding to the armamentarium for combination therapy, and exhibit minimal toxicity, yet have lower manufacturing costs.
HIV virion assembly takes place at the surface membrane of the infected cell where the viral Gag polyprotein accumulates, leading to the assembly of immature virions that bud from the cell surface. Within the virion, Gag is cleaved by the viral proteinase (PR) into the matrix (MA), capsid (CA), nucleocapsid (NC), and C-terminal p6 structural proteins (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)). Gag processing induces a reorganization of the internal virion structure, a process termed “maturation.” In mature HIV particles, MA lines the inner surface of the membrane, while CA forms the conical core which encases the genomic RNA that is complexed with NC. Cleavage and maturation are not required for particle formation but are essential for infectivity (Kohl, N. et al., Proc. Natl. Acad. Sci. USA 85:4686-4690, (1998)).
CA and NC as well as NC and p6 are separated on the Gag polyprotein by short spacer peptides of 14 and 10 amino acids (p2), respectively (spacer peptide 1 (SP1) and SP2, respectively) (Wiegers K. et al., J. Virol. 72:2846-2854 (1998), Pettit, S. C. et al., J. Virol. 68:8017-8027 (1994), Liang et al. J. Virol. 76:11729-11737 (2002)). These spacer peptides are released by PR-mediated cleavages at their N and C termini during particle maturation. The individual cleavage sites on the HIV Gag and Gag-Pol polyproteins are processed at different rates and this sequential processing results in Gag intermediates appearing transiently before the final products. Such intermediates may be important for virion morphogenesis or maturation but do not contribute to the structure of the mature viral particle (Weigers et al. and Pettit, et al., supra). The initial Gag cleavage event occurs at the C terminus of SP1 and separates an N-terminal MA-CA-SP1 intermediate from a C-terminal NC-SP2-p6 intermediate. Subsequent cleavages separating MA from CA-SP1 and NC-SP2 from p6 occur at an approximately 10-fold-lower rate. Cleavage of SP1 from the C terminus of CA is a late event and occurs at a 400-fold-lower rate than cleavage at the SP1-NC site (Weigers et al. and Pettit, et al., supra). The uncleaved CA-SP1 intermediate protein is alternatively termed “p25,” whereas the cleaved CA protein is termed “p24.”
Cleavage of SP1 from the C terminus of CA appears to be one of the last events in the Gag processing cascade and is required for final capsid condensation and formation of mature, infectious viral particles. Electron micrographs of mature virions reveal particles having electron dense conical cores. On the other hand, electron microscopy studies of viral particles defective for CA-SP1 cleavage show particles having a spherical electron-dense ribonucleoprotein core and a crescent-shaped, electron-dense layer located just inside the viral membrane (Weigers et al., supra). Mutations at or near the CA-SP1 cleavage site have been shown inhibit Gag processing and disrupt the normal maturation process, thereby resulting in the production of non-infectious viral particles (Weigers et al., supra). Phenotypically, these particles exhibit a defect in Gag processing (which manifests itself in the presence of a p25 (CA-SP1) band in Western blot analysis) and the aberrant particle morphology described above which results from defective capsid condensation.
Previously, betulinic acid and platanic acid were isolated from Syzigium claviflorum and were determined to have anti-HIV activity. Betulinic acid and platanic acid exhibited inhibitory activity against HIV-1 replication in H9 lymphocyte cells with EC50 values of 1.4 μM and 6.5 μM, respectively, and therapeutic index (T.I.) values of 9.3 and 14, respectively. Hydrogenation of betulinic acid yielded dihydrobetulinic acid, which showed slightly more potent anti-HIV activity with an EC50 value of 0.9 and a T.I. value of 14 (Fujioka, T., et al., J. Nat. Prod. 57:243-247 (1994)). Esterification of betulinic acid with certain substituted acyl groups, such as 3′,3′-dimethylglutaryl and 3′,3′-dimethylsuccinyl groups produced derivatives having enhanced activity (Kashiwada, Y., et al., J. Med. Chem. 39:1016-1017 (1996)). Acylated betulinic acid and dihydrobetulinic acid derivatives that are potent anti-HIV agents are also described in U.S. Pat. No. 5,679,828. Anti-HIV assays indicated that 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid and the dihydrobetulinic acid analog both demonstrated extremely potent anti-HIV activity in acutely infected H9 lymphocytes with EC50 values of less than 1.7×10−5 μM, respectively. These compounds exhibited remarkable T.I. values of more than 970,000 and more than 400,000, respectively.
U.S. Pat. No. 5,468,888 discloses 28-amido derivatives of lupanes
that are described as having a cytoprotecting effect for HIV-infected cells.
Japanese Patent Application No. JP 01 143,832 discloses that betulin and 3,28-diesters thereof are useful in the anti-cancer field.
U.S. Pat. No. 6,172,110 discloses betulinic acid and dihydrobetulin derivatives which have the following formulae or pharmaceutically acceptable salts thereof,

wherein R1 is a C2-C20 substituted or unsubstituted carboxyacyl, R2 is a C2-C20 substituted or unsubstituted carboxyacyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or —OR4, where R4 is hydrogen, C1-4 alkanoyl, benzoyl, or C2-C20 substituted or unsubstituted carboxyacyl; wherein the dashed line represents an optional double bond between C20 and C29.
U.S. Patent Application No. 60/413,451 discloses 3,3-dimethylsuccinyl betulin and is herein incorporated by reference. Zhu, Y-M. et al., Bioorg. Chem. Lett. 11:3115-3118 (2001); Kashiwada Y. et al., J. Nat. Prod. 61:1090-1095 (1998); Kashiwada Y. et al., J. Nat. Prod. 63:1619-1622 (2000); and Kashiwada Y. et al., Chem. Pharm. Bull. 48:1387-1390 (2000) disclose dimethylsuccinyl betulinic acid and dimethylsuccinyl oleanolic acid. Esterification of the 3′ carbon of betulin with succinic acid produced a compound capable of inhibiting HIV-1 activity (Pokrovskii, A. G. et al., Gos. Nauchnyi Tsentr Virusol. Biotekhnol. “Vector,” 9:485-491 (2001)).
Published International Application No. WO 02/26761 discloses the use of betulin and analogs thereof for treating fungal infections.
There exists a need for new HIV inhibition methods that are effective against drug resistant strains of the virus. The strategy of this invention is to provide therapeutic methods and compounds that inhibit the virus in different ways from approved therapies.
The compound and methods of the present invention have a novel mechanism of action and therefore are active against HIV strains that are resistant to current reverse transcriptase and protease inhibitors. As such, this invention offers a completely new approach for treating HIV/AIDS.